Lung Infection Model Research Articles

Human mesenchymal stromal cells inhibit Mycobacterium avium replication in clinically relevant models of lung infection

IntroductionNovel therapeutic strategies are urgently needed for Mycobacterium avium complex pulmonary disease (MAC-PD). Human mesenchymal stromal cells (MSCs) can directly inhibit MAC growth, but their effect on intracellular bacilli is...

An anti-mycobacterial conjugated oligoelectrolyte effective against Mycobacterium abscessus.

Infections caused by nontuberculous mycobacteria have increased more than 50% in the past two decades and more than doubled in the elderly population. Mycobacterium abscessus (Mab), one of the most prevalent of these rapidly growing species, is intrinsically resistant to numerous antibiotics. Current standard-of-care treatments are not satisfactory, with high failure rate and notable adverse effects. We report here a potent anti-Mab compound from the flexible molecular framework afforded by conjugated oligoelectrolytes (COEs). A screen of structurally diverse, noncytotoxic COEs identified a lead compound, COE-PNH2, which was bactericidal against replicating, nonreplicating persisters and intracellular Mab.COE-PNH2 had low propensity for resistance development, with a frequency of resistance below 1.25 × 10-9 and showed no detectable resistance upon serial passaging. Mechanism of action studies were in line with COE-PNH2 affecting the physical and functional integrity of the bacterial envelope and disrupting the mycomembrane and associated essential bioenergetic pathways. Moreover, COE-PNH2 was well-tolerated and efficacious in a mouse model of Mab lung infection. This study highlights desirable in vitro and in vivo potency and safety index of this COE structure, which represents a promising anti-mycobacterial to tackle an unmet medical need.

Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance.IMPORTANCEAcinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.

Prodigiosin as an Antibiofilm Agent against the Bacterial Biofilm-Associated Infection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is known to generate bacterial biofilms that increase antibiotic resistance. With the increase of multi-drug resistance in recent years, the formulation of a new therapeutic strategy has seemed urgent. Preliminary findings show that Prodigiosin (PG), derived from chromium-resistant Serratia marcescens, exhibited efficient anti-biofilm activity against Staphylococcus aureus. However, its anti-biofilm activity against P. aeruginosa remains largely unexplored. The anti-biofilm activity of PG against three clinical single drug-resistant P. aeruginosa was evaluated using crystal violet staining, and the viability of biofilms and planktonic cells were also assessed. A model of chronic lung infection was constructed to test the in vivo antibiofilm activity of PG. The results showed that PG inhibited biofilm formation and effectively inhibited the production of pyocyanin and extracellular polysaccharides in vitro, as well as moderated the expression of interleukins (IL-1β, IL-6, IL-10) and tumor necrosis factor (TNF-α) in vivo, which might be attributed to the downregulation of biofilm-related genes such as algA, pelA, and pslM. These findings suggest that PG could be a potential treatment for drug-resistant P aeruginosa and chronic biofilm infections.

Aerosolized delivery of ESKAPE pathogens for murine pneumonia models

Murine pneumonia models for ESKAPE pathogens serve to evaluate novel antibacterials or to investigate immunological responses. The majority of published models uses intranasal or to a limited extent the intratracheal instillation to challenge animals. In this study, we propose the aerosol delivery of pathogens using a nebulizer. Aerosol delivery typically results in homogeneous distribution of the inoculum in the lungs because of lower particle size. This is of particular importance when compounds are assessed for their pharmacokinetic and pharmacodynamic (PK/PD) relationships as it allows to conduct several analysis with the same sample material. Moreover, aerosol delivery has the advantage that it mimics the ‘natural route’ of respiratory infection. In this short and concise study, we show that aerosol delivery of pathogens resulted in a sustained bacterial burden in the neutropenic lung infection model for five pathogens tested, whereas it gave a similar result in immunocompetent mice for three out of five pathogens. Moreover, a substantial bacterial burden in the lungs was already achieved 2 h post inhalation. Hence, this study constitutes a viable alternative for intranasal administration and a refinement of murine pneumonia models for PK/PD assessments of novel antibacterial compounds allowing to study multiple readouts with the same sample material.

Population pharmacokinetics and humanized dosage regimens matching the peak, area, trough, and range of amikacin plasma concentrations in immune-competent murine bloodstream and lung infection models.

Amikacin is an FDA-approved aminoglycoside antibiotic that is commonly used. However, validated dosage regimens that achieve clinically relevant exposure profiles in mice are lacking. We aimed to design and validate humanized dosage regimens for amikacin in immune-competent murine bloodstream and lung infection models of Acinetobacter baumannii. Plasma and lung epithelial lining fluid (ELF) concentrations after single subcutaneous doses of 1.37, 13.7, and 137 mg/kg of body weight were simultaneously modeled via population pharmacokinetics. Then, humanized amikacin dosage regimens in mice were designed and prospectively validated to match the peak, area, trough, and range of plasma concentration profiles in critically ill patients (clinical dose: 25-30 mg/kg of body weight). The pharmacokinetics of amikacin were linear, with a clearance of 9.93 mL/h in both infection models after a single dose. However, the volume of distribution differed between models, resulting in an elimination half-life of 48 min for the bloodstream and 36 min for the lung model. The drug exposure in ELF was 72.7% compared to that in plasma. After multiple q6h dosing, clearance decreased by ~80% from the first (7.35 mL/h) to the last two dosing intervals (~1.50 mL/h) in the bloodstream model. Likewise, clearance decreased by 41% from 7.44 to 4.39 mL/h in the lung model. The humanized dosage regimens were 117 mg/kg of body weight/day in mice [administered in four fractions 6 h apart (q6h): 61.9%, 18.6%, 11.3%, and 8.21% of total dose] for the bloodstream and 96.7 mg/kg of body weight/day (given q6h as 65.1%, 16.9%, 10.5%, and 7.41%) for the lung model. These validated humanized dosage regimens and population pharmacokinetic models support translational studies with clinically relevant amikacin exposure profiles.

The small RNA PrrH aggravates Pseudomonas aeruginosa-induced acute lung injury by regulating the type III secretion system activator ExsA.

Pseudomonas aeruginosa is a Gram-negative bacterium and the leading cause of nosocomial pneumonia. The pathogenicity of P. aeruginosa is due to the secretion of many virulence factors. Small regulatory RNAs (sRNAs) regulate various bacterial adaptations, especially virulence. Therefore, understanding the mechanism by which sRNAs regulate virulence is necessary for understanding the pathogenicity of P. aeruginosa and the treatment of the related disease. In this study, we demonstrated that PrrH enhances the pathogenicity of P. aeruginosa by binding to the coding sequence regions of the ExsA, the master regulatory protein of type III secretion system, causing severe lung injury and exacerbating the inflammatory response and apoptosis. These findings revealed that PrrH is a crucial molecule that positively regulates ExsA. Type III-positive strains are often associated with a high mortality rate in P. aeruginosa infections in clinical practice. Therefore, this discovery may provide a new target for treating P. aeruginosa infections, especially type III-positive strains.

In Vivo Evolution of a Klebsiella pneumoniae Capsule Defect With wcaJ Mutation Promotes Complement-Mediated Opsonophagocytosis During Recurrent Infection.

Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.

Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.

Pathogenic bacteria and treatment resistance in older cardiovascular disease patients with lung infection and risk prediction model.

This study analyzes the distribution of pathogenic bacteria and their antimicrobial susceptibilities in elderly patients with cardiovascular diseases to identify risk factors for pulmonary infections. A risk prediction model is established, aiming to serve as a clinical tool for early prevention and management of pulmonary infections in this vulnerable population. A total of 600 patients were categorized into infected and uninfected groups. Independent risk factors such as older age, diabetes history, hypoproteinemia, invasive procedures, high cardiac function grade, and a hospital stay of ≥10 days were identified through logistic regression. A predictive model was constructed, with a Hosmer-Lemeshow goodness of fit (P = 0.236) and an area under the receiver operating characteristic curve of 0.795, demonstrating good discriminative ability. The model had 63.40% sensitivity and 82.80% specificity, with a cut-off value of 0.13. Our findings indicate that the risk score model is valid for identifying high-risk groups for pulmonary infection among elderly cardiovascular patients. The study contributes to the early prevention and control of pulmonary infections, potentially reducing infection rates in this vulnerable population.

Differential in vivo labeling with barcoded antibodies allows for simultaneous transcriptomic profiling of airway, lung tissue and intravascular immune cells.

Single-cell RNA sequencing (scRNA-seq) is the state-of-the-art approach to study transcriptomic signatures in individual cells in respiratory health and disease. However, classical scRNA-seq approaches provide no spatial information and are performed using either bronchoalveolar lavage fluid (BAL) or lung single cell suspensions to assess transcript levels in airway and tissue immune cells, respectively. Herein we describe a simple method to simultaneously characterize transcriptomic features of airway, lung parenchymal and intravascular immune cells based on differential in vivo labeling with barcoded antibodies. In addition to gaining basic spatial information, this approach allows for direct comparison of cells within different anatomical compartments. Furthermore, this method provides a time- and cost-effective alternative to classical scRNA-seq where lung and BAL samples are processed individually, reducing animal and reagent use. We demonstrate the feasibility of this approach in a preclinical mouse model of bacterial lung infection comparing airway, parenchymal and vasculature neutrophils early after infection.

Coevolutionary phage training andJoint application delays the emergence ofphage resistance inPseudomonas aeruginosa.

Antibiotic-resistant bacteria are current threats to available antibiotic therapies, and this has renewed interest in the therapeutic use of phage as an alternative. However, development of phage resistance has led to unsuccessful therapeutic outcomes. In the current study, we applied phage training to minimize bacterial phage resistance and to improve treatment outcome by adapting the phage to their target hosts during co-evolution. We isolated and characterized a novel Pseudomonas aeruginosa N4-like lytic phage (PWJ) from wastewater in Yangzhou, China. PWJ is a double-stranded DNA podovirus that can efficiently lyse the model strain ATCC 27,853 and opportunistic pathogen PAO1. Genome sequencing of PWJ revealed features similar to those of the N4-like P. aeruginosa phage YH6. We used PWJ to screen for an evolved trained phage (WJ_Ev14) that restored infectivity to PWJ phage bacterial resisters. BLASTN analysis revealed that WJ_Ev14 is identical to its ancestor PWJ except for the amino acid substitution R1051S in its tail fiber protein. Moreover, phage adsorption tests and transmission electron microscopy of resistant bacteria demonstrated that the R1051S substitution was most likely the reason WJ_Ev14 could re-adsorb and regain infectivity. Furthermore, phage therapy assays in vitro and in a mouse P. aeruginosa lung infection model demonstrated that PWJ treatment resulted in improved clinical results and a reduction in lung bacterial load whereas the joint phage cocktail (PWJ+ WJ_Ev14) was better able to delay the emergence of resister bacteria. The phage cocktail (PWJ +WJ_Ev14) represents a promising candidate for inclusion in phage cocktails developed for clinical applications.

In vitro and in vivo activity of cefiderocol against Achromobacter spp. and Burkholderia cepacia complex, including carbapenem-non-susceptible isolates.

Achromobacter spp. and Burkholderia cepacia complex (Bcc) are rare but diverse opportunistic pathogens associated with serious infections, which are often multidrug resistant. This study compared the in vitro antibacterial activity of the siderophore antibiotic cefiderocol against Achromobacter spp. and Bcc isolates with that of other approved antibacterial drugs, including ceftazidime-avibactam, ciprofloxacin, colistin, imipenem-relebactam, and meropenem-vaborbactam. Isolates were collected in the SIDERO multinational surveillance program. Among 334 Achromobacter spp. isolates [76.6% from respiratory tract infections (RTIs)], cefiderocol had minimum inhibitory concentration (MIC)50/90 of 0.06/0.5 µg/mL overall and 0.5/4 µg/mL against 52 (15.6%) carbapenem-non-susceptible (Carb-NS) isolates. Eleven (3.3%) Achromobacter spp. isolates overall and 6 (11.5%) Carb-NS isolates were not susceptible to cefiderocol. Among 425 Bcc isolates (73.4% from RTIs), cefiderocol had MIC50/90 of ≤0.03/0.5 µg/mL overall and ≤0.03/1 µg/mL against 184 (43.3%) Carb-NS isolates. Twenty-two (5.2%) Bcc isolates overall and 13 (7.1%) Carb-NS isolates were not susceptible to cefiderocol. Cumulative MIC distributions showed cefiderocol to be the most active of the agents tested in vitro against both Achromobacter spp. and Bcc. In a neutropenic murine lung infection model and a humanized pharmacokinetic immunocompetent rat lung infection model, cefiderocol showed significant bactericidal activity against two meropenem-resistant Achromobacter xylosoxidans strains compared with untreated controls (P < 0.05) and vehicle-treated controls (P < 0.05), respectively. Meropenem, piperacillin-tazobactam, ceftazidime, and ciprofloxacin comparators showed no significant activity in these models. The results suggest that cefiderocol could be a possible treatment option for RTIs caused by Achromobacter spp. and Bcc.

Novel Indane-Containing NBTIs with Potent Anti-Gram-Negative Activity and Minimal hERG Inhibition.

Novel bacterial topoisomerase inhibitors (NBTIs) make up a promising new class of antibiotics with the potential to combat the growing threat of antimicrobial resistance. Two key challenges in the development of NBTIs have been to obtain broad spectrum activity against multidrug-resistant Gram-negative bacteria and to diminish inhibition of the hERG cardiac ion channel. Here we report the optimization of a series of NBTIs bearing a novel indane DNA intercalating moiety. The addition of a basic, polar side chain connected to the indane by an ether or an N-linked secondary amide linkage together with a lipophilicity-lowering modification of the enzyme binding moiety led to compounds such as 2a and 2g which showed excellent broad spectrum potency and minimal hERG inhibition. Compound 2a demonstrated robust bactericidal in vivo activity in a mouse lung infection model with the strain P. aeruginosa ATCC 27853 which is resistant to several clinically relevant antibiotics. Rodent pharmacokinetic studies with 2a revealed an unusual profile characterized by rapid tissue distribution and a prolonged, flat terminal phase. This profile precluded further development of these compounds as potential new antibiotics.

Near-infrared light and pH-responsive hyaluronic acid-enveloped ZIF-8 nanoparticles for the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus

There is a serious threat to public health posed by the rapid spread of methicillin-resistant Staphylococcus aureus (MRSA). Photodynamic sterilization is the most effective method of combating bacterial infection, especially multidrug-resistant bacterial infection. In this study, zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, one of the most attractive metal–organic framework material, were used to encapsulate the indocyanine green (ICG, a potent PS); after that, hyaluronic acid (HA) coating was applied to the nanoparticle to create a host cell-tracking nanoplatform (ICG@ZIF-8/HA). Upon near-infrared light irradiation, ICG@ZIF-8/HA exhibit excellent efficacy in producing reactive oxygen species (ROS) and killing multidrug-resistant bacteria in vitro. After the tail vein injection, ICG@ZIF-8/HA can precisely deliver to lung infection sites, and display an effective PDT-based elimination of multidrug-resistant bacteria in mouse lung infection models. These results suggest that the ICG@ZIF-8/HA nanoplatform may exhibit potent curative effects on infectious diseases.

Transcending the challenge of evolving resistance mechanisms in Pseudomonas aeruginosa through β-lactam-enhancer-mechanism-based cefepime/zidebactam.

Compared to other genera of Gram-negative pathogens, Pseudomonas is adept in acquiring complex non-enzymatic and enzymatic resistance mechanisms thus remaining a challenge to even novel antibiotics including recently developed β-lactam and β-lactamase inhibitor combinations. This study shows that the novel β-lactam enhancer approach enables cefepime/zidebactam to overcome both non-enzymatic and enzymatic resistance mechanisms associated with a challenging panel of P. aeruginosa. This study highlights that the β-lactam enhancer mechanism is a promising alternative to the conventional β-lactam/β-lactamase inhibitor approach in combating ever-evolving MDR P. aeruginosa.

Inhibitors of the Elastase LasB for the Treatment of Pseudomonas aeruginosa Lung Infections.

Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa are emerging worldwide as a major threat to human health. Conventional antibiotic monotherapy suffers from rapid resistance development, underlining urgent need for novel treatment concepts. Here, we report on a nontraditional approach to combat P. aeruginosa-derived infections by targeting its main virulence factor, the elastase LasB. We discovered a new chemical class of phosphonates with an outstanding in vitro ADMET and PK profile, auspicious activity both in vitro and in vivo. We established the mode of action through a cocrystal structure of our lead compound with LasB and in several in vitro and ex vivo models. The proof of concept of a combination of our pathoblocker with levofloxacin in a murine neutropenic lung infection model and the reduction of LasB protein levels in blood as a proof of target engagement demonstrate the great potential for use as an adjunctive treatment of lung infections in humans.

Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi.

CRISPR interference (CRISPRi) is a robust gene silencing technique that is ideal for targeting essential and conditionally essential (CE) genes. CRISPRi is especially valuable for investigating gene function in pathogens such as P. aeruginosa where essential and CE genes underlie clinically important phenotypes such as antibiotic susceptibility and virulence. To facilitate the use of CRISPRi in diverse bacteria-including P. aeruginosa-we developed a suite of modular, mobilizable, and integrating vectors we call, "Mobile-CRISPRi." We further optimized Mobile-CRISPRi for use in P. aeruginosa mouse models of acute lung infection by expressing the CRISPRi machinery at low levels constitutively, enabling partial knockdown of essential and CE genes without the need for an exogenous inducer. Here, we describe protocols for creating Mobile-CRISPRi knockdown strains and testing their phenotypes in a mouse pneumonia model of P. aeruginosa infection. In addition, we provide comprehensive guide RNA designs to target genes in common laboratory strains of P. aeruginosa and other Pseudomonas species.

Nuclease activity and protein A release of Staphylococcus aureus clinical isolates determine the virulence in a murine model of acute lung infection.

Staphylococcus aureus is a common cause of hospital-acquired pneumonia associated with high mortality. Adequate clinical treatment is impeded by increasing occurrence of antibiotic resistances. Understanding the underlying mechanisms of its virulence during infections is a prerequisite to finding alternative treatments. Here, we demonstrated that an increased nuclease activity of a S. aureus isolate from a person with cystic fibrosis confers a growth advantage in a model of acute lung infection compared to the isogenic strain with low nuclease activity. Comparing these CF-isolates with a common MRSA-USA300 strain with similarly high nuclease activity but significantly elevated levels of Staphylococcal Protein A (SpA) revealed that infection with USA300 resulted in a significantly increased bacterial burden in a model of murine lung infection. Replenishment with the cell wall-bound SpA of S. aureus, which can also be secreted into the environment and binds to tumor necrosis factor receptor -1 (TNFR-1) to the CF-isolates abrogated these differences. In vitro experiments confirmed significant differences in spa-expression between USA300 compared to CF-isolates, thereby influencing TNFR-1 shedding, L-selectin shedding, and production of reactive oxygen species through activation of ADAM17.

45 Efficacy of the lysin exebacase in addition to vancomycin in a rabbit lung infection model caused by a cystic fibrosis methicillin-resistant Staphylococcus aureus clinical isolate

45 Efficacy of the lysin exebacase in addition to vancomycin in a rabbit lung infection model caused by a cystic fibrosis methicillin-resistant Staphylococcus aureus clinical isolate